Figure - available from: Journal of Tissue Engineering
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Typical confocal images of cardiac tissue samples stained with actinin to identify myocytes, vimentin to identify fibroblasts, and DAPI to identify nuclei. Engineered tissue from (a) nonstimulated and (b) stimulated samples cultured for 12 days. Left ventricular myocardium from (c) P12 and (d) adult rats. Myocytes and fibroblasts are in close spatial proximity. Visually, the (b) stimulated sample has more densely packed myocytes compared to the (a) nonstimulated sample. P12 and adult rat myocardium have densely packed myocytes and fibroblasts. Scale bar: (a) 50 µm applies to all. DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride.
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Quantifying structural features of native myocardium in engineered tissue is essential for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. We applied three-dimensional confocal imaging and image analysis to quantitatively describe the features of native and...
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We previously reported that IKAS are heterogeneously upregulated in failing rabbit ventricles and play an important role in arrhythmogenesis. This study goal is to test the hypothesis that subtype 2 of the small-conductance Ca(2+) activated K(+) (SK2) channel and apamin-sensitive K(+) currents (IKAS) are upregulated in failing human ventricles.
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... However, many of these perfusion cultures are intended for use as in vitro models in developmental biology, safety pharmacology, and drug discovery. Various designs have also been aimed at recreating cardiac-specific biomimetic environments by providing biochemical [47], mechanical [48][49][50], or electrical stimulation [51][52][53], and enhancing nutrient transport [44][45][46]. For example, Roberta et al. designed a bioreactor culture chamber that generates a 3D cardiac structure with bidirectional stromal perfusion and biomimetic electrical stimulation, allowing for direct optical monitoring of cells and contractility testing [54]. ...
Cardiovascular diseases, particularly ischemic heart disease, area leading cause of morbidity and mortality worldwide. Myocardial infarction (MI) results in extensive cardiomyocyte loss, inflammation, extracellular matrix (ECM) degradation, fibrosis, and ultimately, adverse ventricular remodeling associated with impaired heart function. While heart transplantation is the only definitive treatment for end-stage heart failure, donor organ scarcity necessitates the development of alternative therapies. In such cases, methods to promote endogenous tissue regeneration by stimulating growth factor secretion and vascular formation alone are insufficient. Techniques for the creation and transplantation of viable tissues are therefore highly sought after. Approaches to cardiac regeneration range from stem cell injections to epicardial patches and interposition grafts. While numerous preclinical trials have demonstrated the positive effects of tissue transplantation on vasculogenesis and functional recovery, long-term graft survival in large animal models is rare. Adequate vascularization is essential for the survival of transplanted tissues, yet pre-formed microvasculature often fails to achieve sufficient engraftment. Recent studies report success in enhancing cell survival rates in vitro via tissue perfusion. However, the transition of these techniques to in vivo models remains challenging, especially in large animals. This review aims to highlight the evolution of cardiac patch and stem cell therapies for the treatment of cardiovascular disease, identify discrepancies between in vitro and in vivo studies, and discuss critical factors for establishing effective myocardial tissue regeneration in vivo.
... Given the importance of the sarcomeric organization of cardiac tissue, methods for structural characterization are particularly critical to this effort. Scientists and engineers have developed optical methods [2][3][4], electron microscopy (EM) techniques [2,5,6], and other approaches [7][8][9] to characterize sarcomeric organization, but these tools for nanoscale measurement of intact tissue are limited to information from at most a few tens of microns from the surface, resulting in incomplete characterization of the subcellular structure. ...
Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here we use a method not commonly applied to biological materials, small angle x-ray scattering, to characterize the structural development of hiPSC-CMs within three-dimensional engineered tissues during their preliminary stages of maturation. An x-ray scattering experimental method enables the reliable characterization of the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreases as the tissue matures from its initial post-seeding state over the span of 10 days. Visualization of the spacing at a grid of positions in the tissue provides an approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, and disease progression, thereby stimulating new biological hypotheses in stem cell engineering.
... Engineered tissue rings were made of fibrinogen, Matrigel, thrombin and 1-day old Sprague Dawley rat ventricular or atrial CMs. Length, width, height, surface area, and volume of electrically stimulated CMs were not different compared to postnatal rat CMs on day 12. Stimulation increased the expression of CX43 (the membrane is stained positive for Cx43), but Cx43 polarisation was statistically lower than in adult CMs [255]. ...
... Given the importance of the sarcomeric organization of cardiac tissue, methods for structural characterization is particularly important to this effort. Scientists and engineers have developed optical methods [2][3][4] , electron microscopy (EM) techniques 2,5,6 , and other approaches [7][8][9] to characterize sarcomeric organization, but tools for nanoscale measurement of intact tissue are limited to information from at most a few tens of microns from the surface, resulting in incomplete characterization of the subcellular structure. ...
Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here, we used a method not commonly applied to biological materials, small angle X-ray scattering to characterize the structural development of human induced pluripotent stem-cell-derived cardiomyocytes within 3D engineered tissues during their preliminary stages of maturation. An innovative X-ray scattering experimental setup enabled the visualization of a systematic variation in the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreased as the tissue matured from its initial post-seeding state over the span of ten days. Visualization of the spacing at a grid of positions in the tissue provides a new approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, disease progression, thereby stimulating new biological hypotheses in stem cell engineering.
... These results agree with previous studies that ES directs the hECTs toward a phenotype resembling adult-like myocardium. 39 3.4. Intracellular Calcium Imaging in the hECT. ...
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature features, but these are improved by integration into 3D cardiac constructs. In addition, it has been demonstrated that physical manipulations such as electrical stimulation (ES) are highly effective in improving the maturation of human-engineered cardiac tissue (hECT) derived from hiPSC-CMs. Here, we continuously applied an ES in capacitive coupling configuration, which is below the pacing threshold, to millimeter-sized hECTs for 1-2 weeks. Meanwhile, the structural and functional developments of the hECTs were monitored and measured using an array of assays. Of particular note, a nanoscale imaging technique, scanning ion conductance microscopy (SICM), has been used to directly image membrane remodeling of CMs at different locations on the tissue surface. Periodic crest/valley patterns with a distance close to the sarcomere length appeared on the membrane of CMs near the edge of the tissue after ES, suggesting the enhanced transverse tubulation network. The SICM observation is also supported by the fluorescence images of the transverse tubulation network and α-actinin. Correspondingly, essential cardiac functions such as calcium handling and contraction force generation were improved. Our study provides evidence that chronic subthreshold ES can still improve the structural and functional developments of hECTs.
... However, there are still differences between iPSC-CMs and adult CMs, which are characterized by immaturity of cell structure and function, which will lead to fatal arrhythmias after transplantation [13], which is also the main bottleneck in the current treatment of iPSC-CMs. Now, studies have successively revealed methods and strategies to promote the maturation of iPSC-CMs, including prolonging the culture time [14], triiodothyronine [15], dexamethasone (DEX) [16], fatty acids [17,18], electrical stimulation [19] and biomaterials [20], etc. Nonetheless, the underlying mechanisms have not been fully elucidated. We found that DEX was able to inhibit the expression of p-PI3K, p-AKT and p-mTOR in the above induction strategy [21], and the low expression of HIF-1α was associated with the inhibition of the PI3K/AKT/mTOR pathway [22]. ...
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have an irreplaceable role in the treatment of myocardial infarction (MI), which can be injected into the transplanted area with new cardiomyocytes (Cardiomyocytes, CMs), and improve myocardial function. However, the immaturity of the structure and function of iPSC-CMs is the main bottleneck at present. Since collagen participates in the formation of extracellular matrix (ECM), we synthesized nano colloidal gelatin (Gel) with collagen as the main component, and confirmed that the biomaterial has good biocompatibility and is suitable for cellular in vitro growth. Subsequently, we combined the PI3K/AKT/mTOR pathway inhibitor BEZ-235 with Gel and found that the two combined increased the sarcomere length and action potential amplitude (APA) of iPSC-CMs, and improved the Ca²⁺ processing ability, the maturation of mitochondrial morphological structure and metabolic function. Not only that, Gel can also prolong the retention rate of iPSC-CMs in the myocardium and increase the expression of Cx43 and angiogenesis in the transplanted area of mature iPSC-CMs, which also provides a reliable basis for the subsequent treatment of mature iPSC-CMs.
... While standard differentiation protocols do produce cell-to-cell contacts (monolayers and EBs) that facilitate electrical conduction, they are significantly slower compared to the adult heart. hiPSC-CMs are also effectively unloaded, experiencing minimal cell stretch and physiological strain-induced genetic regulation (Lasher et al., 2012;Ruan et al., 2015;Veerman et al., 2015). Thus, several research groups have explored the application of mechanical stress and electrical stimulation, mimicking a more in vivo cellular environment of an adult cardiomyocyte. ...
Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are based on ground-breaking technology that has significantly impacted cardiovascular research. They provide a renewable source of human cardiomyocytes for a variety of applications including in vitro disease modeling and drug toxicity testing. Cardiac calcium regulation plays a critical role in the cardiomyocyte and is often dysregulated in cardiovascular disease. Due to the limited availability of human cardiac tissue, calcium handling and its regulation have most commonly been studied in the context of animal models. hiPSC-CMs can provide unique insights into human physiology and pathophysiology, although a remaining limitation is the relative immaturity of these cells compared to adult cardiomyocytes Therefore, this field is rapidly developing techniques to improve the maturity of hiPSC-CMs, further establishing their place in cardiovascular research. This review briefly covers the basics of cardiomyocyte calcium cycling and hiPSC technology, and will provide a detailed description of our current understanding of calcium in hiPSC-CMs.
... Biphasic ES was firstly considered as an alternative ES mode for reducing the accumulation of by-products in the culture medium resulting by faradaic reactions at the electrode-medium interfaces (Tandon et al., 2009). Preliminary comparative studies showed that biphasic ES induced higher levels of maturation in neonatal rat CMs (Chiu et al., 2011) and in human cardiac progenitor cells compared to monophasic ES (Lasher et al., 2012;Hirt et al., 2014a;Visone et al., 2018a;Visone et al., 2018b;Zhang et al., 2021;Pretorius et al., 2022). However, apart from the two mentioned studies, a clear advantage of biphasic ES was not reported in literature and monophasic ES has been widely adopted in CTE to promote cell maturation (Ronaldson-Bouchard et al., 2018;Zhao et al., 2019). ...
The application of biomimetic physical stimuli replicating the in vivo dynamic microenvironment is crucial for the in vitro development of functional cardiac tissues. In particular, pulsed electrical stimulation (ES) has been shown to improve the functional properties of in vitro cultured cardiomyocytes. However, commercially available electrical stimulators are expensive and cumbersome devices while customized solutions often allow limited parameter tunability, constraining the investigation of different ES protocols. The goal of this study was to develop a versatile compact electrical stimulator (ELETTRA) for biomimetic cardiac tissue engineering approaches, designed for delivering controlled parallelizable ES at a competitive cost. ELETTRA is based on an open-source micro-controller running custom software and is combinable with different cell/tissue culture set-ups, allowing simultaneously testing different ES patterns on multiple samples. In particular, customized culture chambers were appositely designed and manufactured for investigating the influence of monophasic and biphasic pulsed ES on cardiac cell monolayers. Finite element analysis was performed for characterizing the spatial distributions of the electrical field and the current density within the culture chamber. Performance tests confirmed the accuracy, compliance, and reliability of the ES parameters delivered by ELETTRA. Biological tests were performed on neonatal rat cardiac cells, electrically stimulated for 4 days, by comparing, for the first time, the monophasic waveform (electric field = 5 V/cm) to biphasic waveforms by matching either the absolute value of the electric field variation (biphasic ES at ±2.5 V/cm) or the total delivered charge (biphasic ES at ±5 V/cm). Findings suggested that monophasic ES at 5 V/cm and, particularly, charge-balanced biphasic ES at ±5 V/cm were effective in enhancing electrical functionality of stimulated cardiac cells and in promoting synchronous contraction.
... 7−11 Electrical stimulation for enhanced maturation of engineered heart tissues was first performed by Radisic et al. 12 In that study, 8 days of electrical stimulation resulted in cell alignment and coupling along with ultrastructural organization in the engineered cardiac tissues derived from neonatal rat ventricular myocytes (NRVMs). Since then, multiple studies have reported the benefit of electrical pulse stimulation for the enhanced maturation of cardiac tissues derived from either NRVMs 7,8 or hiPSC-CMs. 8−10 However, in these studies, the electrical stimulation was performed through wired systems. ...
In this paper, we report the development of a wireless, passive, biocompatible, and flexible system for stimulation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMS). Fabricated on a transparent parylene/PDMS substrate, the proposed stimulator enables real-time excitation and characterization of hiPSC-CMs cultured on-board. The device comprises a rectenna operating at 2.35 GHz which receives radio frequency (RF) energy from an external transmitter and converts it into DC voltage to deliver monophasic stimulation. The operation of the stimulator was primarily verified by delivering monophasic voltage pulses through gold electrodes to hiPSC-CMs cultured on the Matrigel-coated substrates. Stimulated hiPSC-CMs beat in accordance with the monophasic pulses when delivered at 0.5, 1, and 2 Hz pulsing frequency, while no significant cell death was observed. The wireless stimulator could generate monophasic pulses with an amplitude of 8 V at a distance of 15 mm. These results demonstrated the proposed wireless stimulator's efficacy for providing electrical stimulation to engineered cardiac tissues. The proposed stimulator will have a wide application in tissue engineering where a fully wireless stimulation of electroconductive cells is needed. The device also has potential to be employed as a cardiac stimulator by delivering external stimulation and regulating the contractions of cardiac tissue.
... Given the significant burden of cardiovascular pathology and the shortcomings of 2D systems to recapitulate adult functional benchmarks [18][19][20] , 3D culture systems continue to evolve e.g. [21][22][23][24] , and often make use of concurrent electrical stimulation techniques which have been found to improve CM functionality in vitro [25][26][27] . As induced-pluripotent stem cell (iPSC)-or embryonic stem cell (ESC)-derived CMs can be highly matured when transplanted into adult hearts, logic would dictate that a high degree of in vitro maturation is possible (i.e., there is not a fundamental limit on the level of functional maturation attainable by PSC-CMs), although the conditions necessary to achieve it remain elusive 28 . ...
... Given the importance of retinoids in the heart field and CM subtype specification 59,61 , the continued role of retinoids in evoking higher function would not be unexpected. Mechanical stimuli and progressive electrical pacing 21,24,25,27,62 , and Ca 2+ homeostasis have been demonstrated to be critical in proper CM maturation. Sarcomerogenesis in particular is reliant on mechanical signaling, 63 and sarcomeric complex-mediated mechanical signal transduction may exert significant effects on cellular disposition 64,65 . ...
The development of induced-pluripotent stem cell (iPSC)-derived cell types offers promise for basic science, drug testing, disease modeling, personalized medicine, and translatable cell therapies across many tissue types. However, in practice many iPSC-derived cells have presented as immature in physiological function, and despite efforts to recapitulate adult maturity, most have yet to meet the necessary benchmarks for the intended tissues. Here, we summarize the available state of knowledge surrounding the physiological mechanisms underlying cell maturation in several key tissues. Common signaling consolidators, as well as potential synergies between critical signaling pathways are explored. Finally, current practices in physiologically relevant tissue engineering and experimental design are critically examined, with the goal of integrating greater decision paradigms and frameworks towards achieving efficient maturation strategies, which in turn may produce higher-valued iPSC-derived tissues.
